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Patria 1201, Lomas del Valle, Zapopan, 45110 Jalisco, Mexico. E-mail: [email protected].
Research Paper
Salinity tolerance and other adverse conditions insoils are currently overcome with an excess of irrigationwater, thus given the increasingly shortage of fresh water atglobal level, it is necessary to develop strategies that in-clude both salt-resistant crops and techniques to reducestress injury (Bacilio et al., 2004). A major effort in re-search has been dedicated to organic fertilization or use ofbiofertilizers to increase plant development. Biofertilizersare best described as microbial communities that add, pre-serve and mobilize soil nutrients. Azospirillum is consid-ered one of the most studied plant growth promoting bacte-ria (PGPB) given its ability to significantly increase notonly plant growth development, but also the yield of nu-merous agricultural crops (Givaudan and Bally, 1991;Strzelczyk et al., 1994). Azospirillum brasilense promotesgrowth due to the accumulation and transport of indole-3-acetic acid to the plant (Umali-Garcia et al., 1980; Hart-mann et al., 1983) and other plant growth regulation mole-cules such as absicic acid (ABA) and diamine cadaverine(CAD) (Canto Martín et al., 2004; Perrig et al., 2007).
Azospirillum spp. colonizes the roots of a wide rangeof crops and can efficiently colonize roots submerged ingrowth solutions, and thus increase plant growth. The abil-ity of Azospirillum spp. to stimulate plant growth has beenwidely demonstrated in experiments both field and green-house. Several mechanisms have been suggested to be re-sponsible for the stimulatory effect observed in theinoculated plants in numerous studies and inoculation withAzospirillum spp.
In the last decade it has been demonstrated thatAzospirillum brasilense increases the growth parameters notonly of superior plants but unicellular microalgae Chlorellaspp. by changing its cytology and metabolic parameters (deBashan et al., 2004). This phenomena is also associated withthe potential of Azospirillium spp. to produce plant growthregulators, because the microalga responds to the presenceof Azospirillum similarly to higher plants, increasing theirgrowth and changing its metabolism, due to the greenmicroalgae meet the basic requirements of a higher plant(Mazur et al., 2001; Stirk et al., 2002).
Recently, it has been shown that a recombinantAzospirillum brasilsence Cd strain expressing theomoprotectant trehalose is able to grow in salt stress condi-tions (Rodríguez-Salazar et al., 2009). The aim of this studyis to find out if Chlorella vulgaris, Azospirillum brasilsenceCd and Azospirillum brasilense Cd-BIF, could increase thesalt tolerance of tomato in hydroponic cultures.
Material and Methods
Microalgae and bacteria
Microalgae
Chlorella vulgaris (UTEX 2714) was grown in
axenic cultures of Basal Bold Medium (Bischoff y Holtzer,
1969) at 2000 lux illumination with light/dark periods of
16/8 h for 14 days prior to its use. The cell concentration
was adjusted to 1x106 cells/mL prior to its use as inoculant.
Bacteria
Azospirillum brasilense Cd was grown in nutrient
broth with ampicillin at 30 °C for 72 h prior to its use;
Azospirillum basilense Cd-BIF which enables the accumu-
lation of trehalose in excess (Rodríguez-Salazar et al.,
2009) was grown in nutrient broth supplied with ampicillin
and gentamycin at 30 °C for 72 h prior to its use. The viabil-
ity of the strains was tested on Congo Red agar plates. The
cell concentration was adjusted to 1x109 cells/mL prior to
including a control treatment without microorganisms;
note: combined microbial treatments were tested only in
hydroponic culture in a second set of experiments. Tomato
plants were placed in 13.5 mL of the corresponding media
and 1.5 mL of the corresponding microbial culture (Ta-
ble 1). Plant growth and development was monitored after
10, 20 and 30 days of culture; every period stem and root
length were measured.
Experimental design and statistical analysis
In order to compare the effect of media composition,
time of culture and salt concentration in stem and root elon-
gation, a multifactorial design was used. Data was analyzed
using the ANOVA and Least Significant Difference (LSD)
analysis at level 0.05 of confidence.
Results
Two sets of experiments were planned. In a first
group, two mediums (Hydroponic and Murashige and
Skoog), three periods of time (10, 20 and 30 days), six NaCl
concentrations (0 to 250 mM) and three single cells A.
brasilense Cd (AW), A. brasilense Cd-BIF (AB) and C.
614 Cortés-Jiménez et al.
vulgaris (CV) were analyzed. In a second group, based on
the results of group one, only hydroponic medium was used
given its simplicity and microbial associations were tested
with single or combined free cells.
Stem length
The ANOVA analysis of the first set of experiments
showed that the associated microorganism, elapsed time
and salinity were the only significant factors for plant
growth, despite of the used medium (MS or hydroponic),
(Table 1). It is remarkable that as long as NaCl increased in
concentration form 0 to 250 mM the stem length dimin-
ished, in any case A. brasilense Cd-BIF promoted longer
stems in the seedlings. C. vulgaris had the second better re-
sults in this measure (Figure 1a). Regarding salinity, two
groups are clearly differentiated, Group I: NaCl concentra-
tions 0, 50 and 100 mM with stem lengths average of 22.7 �
0.6 mm and Group II: NaCl concentrations 150, 200 and
250 with stem lengths average of 17.8 � 0.6 mm. When
considering time as a second factor, it can be seen that as
long as time passed the seedlings were less tolerant to salt,
as stem lengths were shorter ~15 and 20 mm in comparison
to the first ten days were stem lengths were in the range of
25 to 26 mm (Figure 1b). In general, those seedlings grow-
ing with any microorganism associated showed longer
stems in comparison to the control, where no microbial as-
sociation was used, which means that salt tolerance is fa-
vored by plant microbial associations.
If the average of stem elongation through the three
periods of time is considered, A. brasilense Cd-BIF showed
the major elongation in the stems (21.8 � 0.07 mm) in com-
parison to the other microbial treatments. However, at the
end of 30 days period the stems were shorter than those ob-
served at 10 days, and this was true for almost all cases, ex-
cept for A. brasilense Cd where the longitudes of the stems
(22.3 � 0.12 mm) were significantly larger (p < 0.05) than
other treatments after 30 days (Figure 1b).
For the second set of experiments, where combined
microorganisms were tested, according to the ANOVA
analysis (Table 2) the combination of A. brasilense Cd and
C. vulgaris (AWCV) had a better impact on stem growth
(25.3 � 0.7 mm) followed by the associated A. brasilense
Cd-BIF and C. vulgaris (ABCV) (23.3 � 0.8 mm) (Figure
2a). Longer stems were observed at 20 days in those seed-
lings associated to A. brasilense Cd and C. vulgaris
(AWCV) (29.8 � 1.3 mm), showing even higher results at
NaCl concentrations below 100 mM (up to 30.6 � 1.7 mm),
but as before, in general the tendency of stems was to de-
crease in size as long as NaCl concentration increased;
nonetheless, seedlings associated with ABCV showed lon-
ger stems than any other treatment at 250 mM of NaCl
(25.7 � 3.3 mM) (Figure 2b).
Root length
Conversely with stem length, the microbial associa-
tion had no statistical significant differences (p < 0.05) in
root length but growing medium (Table 3). Those seedlings
grown in MS medium averaged longer roots (1.29 �
0.03 mM) in contrast to those seedlings grown in hydro-
ponic medium (1.13 � 0.03 mM). Time and salinity also
were significant factors for root length; though, salinity ef-
fects cannot be grouped in two as before (Figure 3a).
Regarding salinity, those seedlings grown in 50 and
100 mM NaCl showed the longer roots, 1.3 � 0.05 and 1.4 �
0.05 mm respectively, in comparison with the rest of the
treatments. In the first 10 days, it can be observed that 50
and 100 mM of NaCl promoted longer roots and this
Associative plant-microbial tolerance 615
Table 1 - Analysis of Variance for Stem Length (mm) for two growing media and simple free cells.
Source SS df MS F-ratio p-value
Main effects
A: Microbial Associa-
tion
919.315 3 306.438 10.69 0.0000
B:Time (d) 1075.01 2 537.505 18.75 0.0000
C:Salinity (mM) 3148.09 5 629.618 21.96 0.0000
D:Medium 39.7511 1 39.7511 1.39 0.2396
Interactions
AB 1628.29 6 271.381 9.46 0.0000
AC 1199.53 15 79.9689 2.79 0.0004
AD 538.267 3 179.422 6.26 0.0004
BC 2292.38 10 229.238 7.99 0.0000
BD 570.949 2 285.475 9.96 0.0001
CD 35.2102 5 7.04204 0.25 0.9419
Residuals 13562.9 473 28.6743
Total (Corrected) 25775.8 525
616 Cortés-Jiménez et al.
Figure 1 - a) Stem lengths for seedlings in relation to NaCl concentrations using different microbial associations for MS and hydroponic media. b) Stem
lengths for seedlings growing in different microbial associations in three periods of time.
Table 2 - Analysis of Variance for Stem Length (mm) for hydroponic media using simple and combined cells.
Source SS df MS F-ratio p-value
Main effects
A: Microbial Associa-
tion
1839.61 5 367.922 13.75 0.0000
B:Time (d) 73.1129 2 36.5565 1.37 0.2565
C:Salinity (mM) 1873.94 5 374.789 14.01 0.0000
Interactions
AB 1878.47 10 187.847 7.02 0.0000
AC 2076.13 25 83.0454 3.1 0.0000
BC 602.975 10 60.2975 2.25 0.0147
Residuals 9419.46 352 26.7598
Total (Corrected) 18329.9 409
growth pattern was consistent for the next 10 days but
showing shorter roots and this is true until 30 days (Figu-
re 3b).
In the second set of experiments where hydroponic
medium was the only one used, time had no statistical effect
on root length, but associated microorganisms and salinity
(Figure 4). According to the analysis of variance, those
Associative plant-microbial tolerance 617
Figure 2 - a) Confidence intervals (� = 0.05) for averaged stem lengths for seedlings growing in association with different microorganisms in hydroponic
medium. b) Stem lengths for seedlings in relation to NaCl concentrations using different microbial associations for hydroponic media in the second set of
experiments.
Table 3 - Analysis of Variance for Root Length (mm) for two growing media.
Source SS df MS F-ratio p-value
Main effects
A: Association 136.578 5 27.3155 2.48 0.0317
B:Time (d) 5.9548 2 2.9774 0.27 0.7633
C:Salinity (mM) 229.184 5 45.8369 4.16 0.0011
Interactions
AB 737.614 10 73.7614 6.7 0
AC 531.986 25 21.2795 1.93 0.0053
BC 412.965 10 41.2965 3.75 0.0001
Residuals 3888.76 353 11.0163
Total (Corrected) 5785.52 410
seedlings associated with C. vulgaris where different to
control having longer roots (12 � 0.4 mm) (Figure 4a).
Even when the effects of NaCl concentration are not differ-
entiated in groups as easily as with stems, in roots is note-
worthy the influence of 100 mM of NaCl with longer roots
(12.6 � 0.4 mm).
Concomitantly, AW had a marked effect on root elon-
gation after 30 days (13.2 � 0.7 mm), similar to that ob-
served for CV at the end of the first 10 days (13.2 � 0.8 mm)
with control showing the shorter roots after 20 and 30 days
of treatment (Figure 4b).
Discussion
Hydroponic culture of plants with sea water or even
salted water and the use of biofertilizers is a promising
technology in order to mitigate the salinity effect on food
crops. In the present work we planned to analyze the effects
of Azospirillum brasilense Cd wild type, A. brasilense
Cd-BIF able to over accumulate the osmoprotectant
trehalose and Chlorella vulgaris in the growth of tomato
seedlings using two different culture media and in micro-
bial association.
According to the observed results, Azospirillum
basilense Cd-BIF followed by Chlorella vulgaris showed
the better influence in seedlings promoting longer stems,
with good results even at 250 mM of NaCl. Given that mi-
crobial associations showed in general better results for
stem and roots elongation in tomato seedlings, this could
imply that these plants could be irrigated with sea water at
least partially when associated to any of this microorgan-
isms. Seemingly, if the concentration of NaCl is increased
for those seedlings growing in hydroponic medium, the ef-
fect of A. brasilense Cd-BIF will be better; meanwhile the
triple association of A. brasilense Cd, C. vulgaris plus seed-
lings appears to be sensitive to NaCl increments. These re-
sults are in agreement with those works reported previously
about Azospirillum brasilense growing up to 200 mM of
NaCl, where no drop in bacterial growth rate was observed,
but when 300 mM of NaCl was used, the growth rate dimin-
ished in 66% (Rivarola et al., 1998). In our results, A.
brasilense Cd showed almost the same growing behavior,
618 Cortés-Jiménez et al.
Figure 3 - a) Confidence intervals (� = 0.05) for averaged stem lengths for seedlings growing at different NaCl concentrations in MS and hydroponic me-
dia. b) Effect of salinity concentration on root length through the time for MS and hydroponic media.
however we assume that A. brasilense Cd-BIF could grow
with no problems even up to 300 mM.
In previous works with plants of Zea mayz under os-
motic stress, Rodríguez-Salazar et al. (2009) observed an
increased biomass, namely thicker roots, of those plants
grown in association with A. brasilense Cd-BIF. Other
works with maize and wheat report an osmoadaptative phe-
nomena for those plant cells associated with A. brasilense
(El-Baky et al., 2008)(2, 16). Some other examples in
wheat and barley suggest that the association plant-
microorganism are beneficial for plant’s salt tolerance due
to an increase in its growing capacity (Caballero-Mellado,
2006; Zawoznik et al., 2011).
On the other hand, Hiremath and Mathad et al. (2010)
demonstrated that Chlorella vulgaris was positively stimu-
lated by up to 200 mM of NaCl for chlorophyll and proline
production, but when salinity concentration was increased
up to 300 mM a reduction on chlorophyll was observed.
Díaz et al. (1999)) reported that proline accumulates in
plants in response to increased environmental salinity.
These supports the evidence showed here regarding C.
vulgaris and its effect on plant tolerance as mentioned
above, where C. vulgaris had the second marked effect on
salt tolerance over the seedlings of tomato.
In general, stems and roots lengths decreased with
time, despite of the applied treatment and this could be a
cause of the prolonged immersion time of the seedlings in
the nutritive solutions, thus is advisable to add some kind of
support for the plant. This is why for a future work we are
planning to evaluate the effect of these microbial species in
seedlings growing in alginate beads supports. As our results
suggests, the use of A. brasilense Cd-BIF and C. vulgaris is
a viable approach to increase the salt tolerance in plants and
their biomass and the possible use of sea water to irrigate
horticultural plants.
Associative plant-microbial tolerance 619
Figure 4 - a) Root lengths for seedlings in relation to NaCl concentrations using different microbial associations for hydroponic media in the second set
of experiments. b) Root lengths for seedlings growing in different microbial associations for three periods of time in hydroponic media in the second set of
experiments.
Acknowledgement
The main authors wish to thank the Doctoral fellow-
ship 162578 by CONACYT to Daniel Cortés and Abril
Gómez, respectively. We express our gratitude to Virginia
Berenice Suarez for her English correction proof.
Abbreviations
AW, Azospirillum brasilense Cd (wild type)
AB, Azospirillum brasilense Cd with plasmid
pBBR1M:BIF
PGPB, Plant Growth Promoting Bacteria
CV, Chlorella vulgaris
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